Genomic and Physiological Characteristics of a Novel Nitrite-OxidizingNitrospiraStrain Isolated From a Drinking Water Treatment Plant

Nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, which is an important process of the biogeochemical nitrogen cycle and is exploited extensively as a biological nitrogen removal process. Members of the genusNitrospiraare often identified as the dominant NOB in a diverse range of natural and artificial environments. Additionally, a number of studies examining the distribution, abundance, and characterization of complete ammonia oxidation (comammox)Nitrospirasupport the ecological importance of the genusNitrospira. However, niche differentiation between nitrite-oxidizingNitrospiraand comammoxNitrospiraremains unknown due to a lack of pure cultures. In this study, we report the isolation, physiology, and genome of a novel nitrite-oxidizingNitrospirastrain isolated from a fixed-bed column at a drinking water treatment plant. Continuous feeding of ammonia led to the enrichment ofNitrospira-like cells, as well as members of ammonia-oxidizing genusNitrosomonas. Subsequently, a microcolony sorting technique was used to isolate a novel nitrite-oxidizingNitrospirastrain. Sequences of strains showing the growth of microcolonies in microtiter plates were checked. Consequently, the most abundant operational taxonomic unit (OTU) exhibited high sequence similarity withNitrospira japonica(98%) at the 16S rRNA gene level. The two otherNitrospiraOTUs shared over 99% sequence similarities withN. japonicaandNitrospirasp. strain GC86. Only one strain identified asNitrospirawas successfully subcultivated and designated asNitrospirasp. strain KM1 with high sequence similarity withN. japonica(98%). The half saturation constant for nitrite and the maximum nitrite oxidation rate of strain KM1 were orders of magnitude lower than the published data of other knownNitrospirastrains; moreover, strain KM1 was more sensitive to free ammonia compared with previously isolatedNitrospirastrains. Therefore, the newNitrospirastrain appears to be better adapted to oligotrophic environments compared with other known non-marine nitrite oxidizers. The complete genome of strain KM1 was 4,509,223 bp in length and contained 4,318 predicted coding sequences. Average nucleotide identities between strain KM1 and known culturedNitrospiragenome sequences are 76.7-78.4%, suggesting at least species-level novelty of the strain in theNitrospiralineage II. These findings broaden knowledge of the ecophysiological diversity of nitrite-oxidizingNitrospira.